A simple reverse transcription loop–mediated isothermal amplification combined with visual detection method (vRT–LAMP) assay was developed for rapid and specific detection of porcine epidemic diarrhea virus (PEDV) in this study, which overcomes the shortcomings of previously described RT–LAMP assays that require additional detection steps or pose a risk of cross-contamination. The assay results can be directly detected by the naked eye using hydroxynaphthol blue after incubating for 40 min at 62 °C. The assay specifically amplified PEDV RNA and no other viral nucleic acids. The limit of detection of the assay was less than 50 RNA copies per reaction, which was 100 times more sensitive than conventional reverse transcription polymerase chain reaction (RT-PCR) and comparable to real-time RT-PCR (RRT-PCR). In the clinical evaluation, the PEDV detection rate of vRT–LAMP was higher than that of RRT-PCR, showing 99 % concordance, with a kappa value (95 % confidence interval) of 0.97 (0.93–1.01). Considering the advantages of high sensitivity and specificity, simple and direct visual monitoring of the results, no possibility for cross–contamination, and being able to be used as low-cost equipment, the developed vRT–LAMP assay will be a valuable tool for detecting PEDV from clinical samples, even in resource-limited laboratories.

本研究开发了一种简单的逆转录环介导的等温扩增结合视觉检测方法（vRT-LAMP）测定法，用于快速特异性检测猪流行性腹泻病毒（PEDV），克服了先前描述的RT-LAMP测定法的缺点需要额外的检测步骤或构成交叉污染的风险。使用羟基萘酚蓝在 62°C 下孵育 40 分钟后，可以直接用肉眼检测测定结果。该测定特异性扩增PEDV RNA，没有其他病毒核酸。该测定的检测限是每个反应少于 50 个 RNA 拷贝，比传统的逆转录聚合酶链式反应 (RT-PCR) 灵敏 100 倍，与实时 RT-PCR (RRT-PCR) 相当。在临床评价中，vRT-LAMP 的 PEDV 检出率高于 RRT-PCR，一致性为 99%，kappa 值（95% 置信区间）为 0.97（0.93-1.01）。考虑到高灵敏度和特异性、简单直接的结果可视化监测、没有交叉污染的可能性以及能够用作低成本设备的优点，开发的 vRT-LAMP 检测将成为一种有价值的检测工具来自临床样本的 PEDV，即使在资源有限的实验室。